肿瘤诱导CAF长非编码RNA CAFUL1表达上调促进肺腺癌侵袭转移的机制研究

CAFUL1 is a cancer associated fibroblasts (CAFs) specific up-regulated lncRNA which is scanned from high-throughput sequencing of CAFs derived from lung adenocarcinoma (LUAD) and TCGA datasets. We found that CAFUL1 up-regulated specifically in CAFs, and a higher expression level of CAFUL1 is associated with LUAD metastasis and poorer survival. Silencing CAFUL1 in CAFs could suppress the invasion and metastasis of LUAD cells. The present preliminary data suggested that CAFUL1 could activate IKKβ phosphorylation and downstream NF-κB signaling pathway and subsequently promote LUAD invasion and metastasis. Additionally, LUAD cells could secrete IL-18 so as to activate c-MYC and stimulate the down stream transcription of CAFUL1. In summary, we proposed that LUAD cells secrete IL-18 to up-regulate CAFUL1 expression in CAFs, which activating NF-κB signaling pathway and eventually leading LUAD invasion and metastasis. In the present project, we aimed to confirm that CAFs with CAFUL1 up-regulating could promote LUAD metastasis at first, and then clarify the crosstalk mechanism between LUAD cells and CAFs based on IL18/c-MYC/CAFUL1/IKKβ/NF-κB pathway. Finally, we will evaluate the clinical relevance of CAFUL1, as well as which upstream and downstream genes,based on clinical samples. Our research is the original source of innovation and can provide new potential bio-markers for the prevention and treatment of lung cancer.

CAFUL1（简称CL）是项目组基于肺腺癌原代肿瘤相关成纤维细胞（CAF）高通量测序及TCGA大数据筛选出的一条CAF特异性高表达、功能未知lncRNA。前期发现：CL仅高表达于CAF，正常组织及肿瘤细胞几乎不表达；CL表达与转移正相关，表达越高预后越差；沉默CAF中CL则共培养肺腺癌细胞迁移能力降低。生信分析及预实验示：CAF中CL可激活IKKβ磷酸化及下游NF-κB通路，肺腺癌可释放IL18激活CAF中MYC通路促进CL转录。故提出肺腺癌释放IL18促进CAF中CL转录、进而激活NF-κB通路、促进肺腺癌转移的科学假说。本项目拟体内外明确CL高表达CAF可促进肺腺癌转移；设计拯救实验，阐明IL18/CL/NF-κB介导的肺腺癌/CAF对话机制；临床大样本评价CL上下游基因与肺腺癌转移的相关性。有关CAF特异性高表达CL介导的肺腺癌/CAF对话机制未见报道，本研究可为肺癌防治提供新思路。

肺癌发病率及死亡率一直稳居所有恶性肿瘤之首，其中超过2/3病理类型为肺腺癌（Lung adenocarcinoma，LUAD）。深入挖掘肺腺癌侵袭转移这一重要临床难题背后的分子机制一直是该领域研究热点。自后基因组时代以来，转录组学一直是肿瘤领域研究前沿。. 对于在肺腺癌组织及硬基质来源的肿瘤相关成纤维细胞（stCAFs）中高表达的长链非编码RNA CAFUL1，课题组结合泛肿瘤基因组学和生物机械力研究，对CAFUL1的生物学特性进行了深入的探索。项目组通过对本院肺腺癌组织行天狼星红染色和原子力显微镜法测量机械力，将组织分为基质沉积型和基质松散型，再结合CAFs相关基因的表达高低将两型肺腺癌组织包含的CAFs分别定义为硬基质来源CAFs（stCAFs）和软基质来源CAFs（soCAFs）。通过对软硬基质来源CAFs的转库组测序结合TCGA、CCLE第三方数据库筛选发现CAFUL1是一个肺腺癌中高表达、与基质硬度呈正相关的lncRNA，并通过肺腺癌组织验证并表明其是肺腺癌预后不良的独立预测因素。体内、外功能实验表明CAFUL1促进基质沉积和细胞-基质粘附从而促进肺腺癌侵袭进展。后续项目实施过程中，发现stCAFs中高表达的CAFUL1促进ROCK1介导的MLC2磷酸化和胶原收缩，从而介导细胞外基质重塑，使细胞间粘附增加，限制了疗效从而导致肺腺癌的恶性进展。. 本项目对肺腺癌中stCAFs高表达lncRNA的表达水平、基因组扩增水平及临床预后的关系进行了深入的分析，鉴定出CAFUL1等多个潜在的肺腺癌驱动lncRNA。并首次对CAFUL1的分子作用机制进行了深入的研究，证实其在细胞质中结合ROCK1和MLC2，以ROCK1依赖的方式促进MLC2磷酸化，在转录后水平介导肌球蛋白收缩和细胞-基质粘附从而促进肺腺癌侵袭的分子机制。本研究具有源头创新性，从lncRNA的角度为开发肺腺癌核酸治疗药物提供潜在的新靶标。